A generic microfluidic biosensor of G protein-coupled receptor activation – impedance measurements of reversible morphological changes of reverse transfected HEK293 cells on microelectrodes

Abstract

Impedance spectroscopy of cell lines on interdigitated electrodes (IDEs) is an established method of monitoring receptor-specific cell shape changes in response to certain analytes. Normally, assays are done in multiwells making it a bulky, static and single use procedure. Here, we present a biosensor allowing sequential application of biological test samples with an automated microfluidic system. It is capable of monitoring relative changes in impedance using castellated IDEs of 250–500 μm diameter, covered with stable or reverse transfected HEK293 cells. Reversible activation of the Neurokinin 1 (NK1) receptor in stable cell lines was observed in response to a series of 5 minute exposures from 1 pM–10 nM of the specific ligand Substance P (SP) using impedance measurements at 10 mV and 15 kHz. An optimal flow speed of 10 μl min⁻¹ was chosen for the 10 μl flow cell. The EC₅₀ of ∼10 pM was about 10 times lower than the EC₅₀ based on measuring changes in the calcium ion concentration. The method was also shown to work with reverse transfected cells. Plasmid DNA encoding the NK1 gene was spotted onto the electrodes and pre-incubated with a transfection agent. The overlaid HEK293 cells were subsequently transfected by the underlying DNA. After challenge with SP, the cells induced an activation response similar to the stable cell line. The microfluidic micro-electrode reverse transfection system opens up possibilities to perform parallel measurements on IDE arrays with distinct receptors per IDE in a single flow channel.